Since the earliest descriptions of the electrocardiogram, the period of quiescence between each depolarization, has fascinated scientists. This phase of the cardiac cycle is now known to be a highly orchestrated series of events that restore the resting membrane potential. Repolarization is a complex phenomenon, dependent on the functions of individual channels, receptors, cytoskeleton and the membrane, all within a multicellular structure. In the last decade, perturbations of repolarization have been directly implicated in fatal cardiac arrhythmias. Given this sensitivity to aberrations of repolarization, it is not surprising that there are robust mechanisms defending this process. One of the central hypotheses in the field is that the heart has a repolarization reserve that allows it to tolerate small perturbatations of repolarization, but which can be overwhelmed by severe or cumulative insults. Given the complexity of this process, an integrated whole animal model organism amenable to rapid genetic manipulation and analysis will be required for a better understanding of repolarization. Our preliminary data suggest that disturbances of repolarization are manifest as bradycardia in the zebrafish, and that this organism will provide a good model for the analysis of cardiac repolarization. This proposal takes a systematic approach to the study of cardiac repolarization in the zebrafish in three specific aims: 1) establish the mechanism of bradycardia in response to lKr blocking drugs in the zebrafish; 2) characterize the zebrafish orthologs of the human long QT syndrome genes and assess their role in zebrafish cardiac repolarization; 3) systematically test the concept of repolarization reserve by modulating the molecular determinants of repolarization in the zebrafish. The candidate has a background in chemistry and molecular biology and has recently completed cardiology and electrophysiology fellowship training. Dr. Milan was appointed to the staff of the Cardiology Division at MGH in July 2003, and the Division has committed numerous resources to his work. He has assembled a group of three sponsors who will guide him in both zebrafish biology and genetics and cardiovascular cellular physiology. Dr. Milan's panel of advisors includes leaders in the fields of cardiac repolarization, cardiomyocyte signal transduction and drug-induced cardiac arrhythmias. Dr. Milan will complement the experimental work described in this proposal with a comprehensive educational program including lab meetings, journal clubs, seminars, scientific meetings, and formal coursework. This plan is carefully designed to result in Dr. Milan's progressive scientific, professional and personal development, culminating in his emergence as an independent investigator in the field of basic cardiac electrophysiology.